UCSF

The hypothesis of my thesis project was that targeting the extracellular matrix of a solid tumor for a local generation of a cytotoxic drug could inhibit tumor growth and progression. To test this hypothesis of the matrix attachment therapy (MAT), a gene therapy and recombinant protein therapy were pursued. For the gene therapy, I cloned a mammalian expression vector containing the extracellular domain of CD44 (sCD44), yeast cytosine deaminase (CD), and E.coli uracil phosphoribosyltransferase (UPRT) genes to transfect tumor cells in vitro. The sCD44::CD::UPRT fusion gene construct was designed to secrete the expressed fusion protein and generate cytotoxic drugs to cause cell deaths in the presence of the prodrug 5-fluorocytosine (5-FC). This gene therapy strategy did not result in a potent cytotoxic effect on cells that expressed the fusion gene due to low expression of the fusion genes in transfected cells.

For the protein therapy, a recombinant fusion protein containing TSG-6 Link (Link) and the CD enzyme was expressed in E.coli and purified to characterize the functions of the LinkCD fusion protein. LinkCD exhibits Km of 0.33 mM and Vmax of 14 µM/min/µg for the conversion of 5-FC to 5-FU. LinkCD can bind to a hyaluronan oligomer (12-mer) at a KD of 55 µM at pH 7.4 and a KD of 5.32 µM at pH 6.0 measured using surface plasmon resonance (SPR). To evaluate the anti-tumor effect of LinkCD/5-FC combination therapy in vivo, mice received intratumoral injections of LinkCD on days 11 and 14 after C26 tumor implantation and drinking water containing 10 mg/mL of 5-FC starting on day 11. Animals that received LinkCD/5-FC treatment showed significant tumor size reduction and increased survival compared to the CD/5-FC treatment group. Other treatment groups that were unable to produce 5-FU had no effect on the tumor growth despite receiving the fusion protein that contained the Link domain. The results strongly suggest that a treatment regime consisting of a fusion protein containing the Link domain, the active CD enzyme, and the prodrug 5-FC are required to produce an anti-tumor effect. Intravenous administration of LinkCD to animals tumored with C26 that received 5-FC in the drinking water did not result in a therapeutic effect, possibly due to a short circulation time of LinkCD. I created chimeras of the CD44 Link domain in silico that could potentially be used to increase the circulation time of LinkCD in the blood. Thus, MAT is a promising alternative to antibody-directed prodrug enzyme therapy approach for cancer treatment.